The primary goal of the proposed studies genetically dissect the mechanism of nonself recognition in filamentous fungi which leads to fungal programmed cell death (PCD) using Neurospora crassa as a model system. Nonself recognition is essential for defense against pathogen invasion and prevention of resource plundering. Our hypothesis is that filamentous fungi, such as Neurospora crassa, have harnessed protein heterocomplex formation as a mechanism to accomplish nonself recognition, which leads to fungal PCD. Nonself recognition during vegetative growth in filamentous fungi is mediated by genetic loci, termed het (for heterokaryon) loci, which regulate the viability of heterokaryons that occur as a result of hyphal fusion between genetically different individuals, het loci show evidence of balancing selection, similar to loci within the Major Histocompatibility Complex (MHC) which are part of the mammalian immune system. Heterokaryon incompatibility is a form of programmed cell death, which can be defined as an orderly process in which a cell actively participates in its own death. Heterokaryon incompatibility is a fungal "immune" system that confers selective advantages to filamentous fungi by preventing hyphal fusion events that spread mycoviruses, debilitated organelles and deleterious plasmids throughout a fungal population. Our initial results (supported by our previous NIH grant) show that nonself recognition occurs by formation of a HET-C protein heterocomplex associated with the plasma membrane, which appears to be fundamentally different in stability/structure than HET-C monomers. Our data suggests that HET-C interacts with a second protein carrying a HET domain; HET domain genes are common, but of unknown function, in filamentous fungal genomes, including human fungal pathogens. This protein complex formation activates signal transduction mechanisms and gene regulation, ultimately resulting in death and destruction of the heterokaryotic cell. The experiments described in this proposal aim to further investigate the role the HET-C/VIB-2 heterocomplex in nonself recognition, and MAP kinase pathways and the VIB-1 transcriptional regulator in triggering fungal PCD. We believe that a dissection of fungal PCD in N. crassa will illuminate conserved and divergent features of PCD among eukaryotic organisms, provide potential drug targets for pathogenic filamentous fungi.